The neuropeptide slow transmitters PACAP (pituitary adenylate cyclase activating polypeptide) and arginine vasopression (AVP) are released at synapses that transduce stress responses to the brain, and mediate homeostatic and allostatic adjustments to stress by the organism. The catecholamine transmitter DA (dopamine) is released at synapses that transduce reward and mediate response to psychomotor stimulant drugs of abuse. Allostatic responses to systemic and psychogenic stressors, shown to be PACAP-dependent, are implicated as causative factors in depression and post-traumatic stress disorder (PTSD). Stress is implicated as a driver of both decreased reward drive (anhedonia), and of anxious behavior, both contributory to depression, PTSD, and generalized anxiety disorder (GAD) occurring in humans. DA neurotransmission is thought to underlie organismic drive for reward, as well as to mediate both the rewarding properties of psychomotor stimulants, and the likelihood of relapse to drug-seeking behavior that can be triggered by stress. Thus, these two systems (DAergic and PACAPergic) are likely to strongly interact. The extrahypothalamic projections of vasopressinergic magnocellular neurons of the paraventricular nucleus (PVN) are also being explored as modulators of stress and sex-hormone-dependent inputs to amygdala and lateral habenula (LHb) that control motor responses (including exploration and escape) to threat-related stressful stimuli. Understanding the cellular mechanisms of stress transduction, and stress interaction with reward circuitry, at the circuit, cellular and molecular levels is crucial to developing effective therapeutic interventions for these disorders. PACAP, DA, and AVP act through Gs- or Gq-coupled G-protein receptors (GPCRs) on target cells (neurons) of the brain, exerting their actions as first through a precisely time-dependent elevation of intracellular cyclic AMP (cAMP) or other second messengers including calcium. For Gs-coupled signaling via cAMP, we have identified distinct contributions of the three major cAMP sensors PKA, Epac (Rapgefs 3 and 4), and NCS/Rapgef2 to cAMP-dependent processes carried out by NS-1 neuroendocrine cells and primary neurons in cellula (A.C. Emery, M.V. Eiden and L.E. Eiden, J.Biol. Chem. 289: 10126, 2014; A.C. Emery et al., ACS Chemical Neuroscience DOI: 10.102/acschemneuro.6b00462; A.C. Emery et al., J. Biol. Chem.DOI: 10.1074/jbc.M117.790329). Either of the two first messengers, PACAP and DA, alluded to above as important in stress transduction and in reward, activate these three 'parcellated' cAMP-dependent pathways. The relative contributions of the three cAMP-dependent pathways to the actions of PACAP and DA at the specific post-synaptic sites that characterize the stress response and reward micro-circuitry of the mammalian CNS are under investigation. How these intracellular mechanisms determine the interaction between PACAPergic and DAergic neurotransmission that ultimately leads to the depressogenic and anxiogenic effects of stress, the rewarding characteristics of psychomotor stimulants, and the stress x reward interactions that blunt hedonia and increase anxiety in stress-associated affective disorders, and in stress-associated relapse to drug-seeking behavior are being studied in mice in which components of these signaling systems are deleted in specific brain areas. We are focusing on the relative contributions of the three sensors to cAMP-dependent effects mediated by the Gs-coupled PACAP receptor PAC1, and the dopamine D1 receptor, in the extended amygdala and in the ventral striatum, respectively, during the processes of development of chronic stress-induced anxious/depressive behavior, and psychomotor stimulant-induced plasticity of medium spiny neurons, respectively. These studies now involve examination, using neurochemical, histochemical, and tracing methods, micro-circuitry of PACAPergic innervation in both extended amygdala and in the ventral striatum. Although PACAPergic cell bodies, and PACAPergic nerve terminals have been extensively characterized in the rodent brain, the connections between them (i.e. the circuits in which they function, including those modulating reward and anxiety) remain largely uncharacterized, as do their interactions with dopaminergic reward circuits in ventral striatum. We have recently determined that PACAP-dependent effects on corticosterone secretion in response to a single episode of restraint stress (at the level of PACAPergic innervation of CRH neurons of the paraventricular nucleus of the hypothalamus, i.e. hypothalamo-pituitary-adrenal, or HPA axis activation) can be dissociated from PACAP-dependent effects on behavior (hypophagia) associated with this stressor (S. Z. Jiang et al., Stress 19: 374, 2016). These results imply that PACAPergic circuitry underlying HPA axis activation is separate from PACAPergic circuitry underlying suppression of feeding (stress-associated anorexia), and that post-synaptic PACAP effects on CRH neurons (affecting long-term CRH synthesis without affecting immediate CRH secretion) must be fundamentally different from post-synaptic PACAP effects on the neurons mediating acute restraint stress-dependent effects on feeding. This work forms the basis for our continuing efforts to define PACAPergic functional circuits involved in stress responding and in modulation of DAergic neurotransmission relevant to reward, and the cAMP-dependent and -independent post-synaptic intracellular signaling pathways that are finally responsible for the physiological regulation of stress responding by these two neurotransmitters in the brain. An additional project related to GPCR ligand-mediated stress responding in the brain, in collaboration with Dr. Limei Zhang-deBarrio, visiting Fulbright Scholar, UNAM, Mexico, involves identification and tracing of vasopressinergic magnocellular neurons projecting to amygdala and habenula, and linking anxiety and escape behavior response modification by environmental and homeostatic cues including thirst and sex hormone status in the behaving rat. These studies extend the concept that neuropeptides functioning as hormones upon secretion from the hypothalamus for homeostatic regulation also coordinate allostatic responses to the environment through their projections to extrahypothalamic brain( V. S. Hernndez et al., Frontiers in Neuroal Circuits DOI: 10.3389/fncir.2016.00092; and L. Zhang et al., in preparation).